Over the past decade, transgenic plants have been successfully used to express a variety of proteins in plants, including genes from bacterial and viral pathogens. Numerous genes have been cloned into a variety of transgenic plants including many enzymes that have demonstrated the same enzymatic activity as their authentic counterparts. See, for example, expression of avidin in plants, U.S. Pat. No. 5,767,379; aprotinin expressed in plants, U.S. Pat. No. 5,824,870 and proteases expressed in plants, U.S. Pat. No. 6,087,558.; Hood, E. E., D. R. Withcher, S. Maddock, T. Meyer, C. B. M. Baszczynski, M. Bailey, P. Flynn, J. Register, L. Marshal, D. Bond, E. Kulisek, A. Kusnadi, R. Evangelista, Z. Nikolov, C. Wooge, R. J. Mehigh, R. Heman, W. K. Kappel, D. Ritland, P. C. Li, and J. A. Howard, 1997, Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Molecular Breeding 3:291-306; Pen, J., L. Molendijk, W. J. Quax, P. C. Sijmons, A. J. van Ooyen, P. J. van den Elzen, K. Rietveld, and A. Hoekema, 1992, Production of active Bacillus licheniformis α-amylase in tobacco and its application in starch liquefaction. Biotechnology 10:292-296; Trudel, J., C. Potvin, and A. Asselin 1992 Expression of active hen egg white lysozyme in transgenic tobacco. Plant Sci. 87:55-67.
Many additional genes have been expressed in plants solely for their immunogenic potential, including viral proteins (U.S. Pat. No. 6,136,320; Mason, H. S., J. M. Ball, J.-J. Shi, X. Jiang, M. K. Estes, and C. J. Arntzen. 1996. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc. Natl. Acad. Sci. USA 93:5335-5340; Wigdorovitz, supra; Kapusta, et al, supra; McGarvey, P. B., J. Hammond, M. M. Dienelt, D. C. Hooper, Z. F. Fu, B. Dietzschold, H. Koprowski, and F. H. Michaels. 1995. Expression of the rabies virus glycoprotein in transgenic tomatoes. Biotechnology 13:1484-1487; Thanavala, Y., Y.-F. Yang, P. Lyons, H. S. Mason, and C. J. Arntzen. 1995. Immunogenicity of transgenic plant-derived hepatitis B surface antigen. Proc. Natl. Acad. Sci. USA 92:3358-3361) and subunits of bacterial toxins (Arakawa, T., D. K. X. Chong, J. L. Merritt, W. H. R. Langridge. 1997. Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res. 6:403-413; Arakawa, T., J. Yu, and W. H. Langridge. 1999. Food plant-delivered cholera toxin B subunit for vaccination and immunotolerization. Adv. Exp. Med. Biol. 464:161-178; Haq, T. A., H. S. Mason, J. D. Clements, and C. J. Arntzen. 1995. Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268:714-716). Animal and human immunization studies have demonstrated the effectiveness of many plant derived recombinant antigens in stimulating the immune system. The production of antigen-specific antibodies and protection against subsequent toxin or pathogen challenge demonstrates the feasibility of plant derived-antigens for immunologic use. For example, the resulting peptides induced an immunogenic response in mice (Mason, H. S., T. A. Haq, J. D. Clements, C. J. Arntzen. 1998. Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine 16:1336-1343; Wigdorovitz, A., C. Carrillo, M. J. Dus Santos, K. Trono, A. Peralta, M. C. Gomez, R. D. Rios, P. M. Franzone, A. M. Sadir, J. M. Escribano, M. V. Borca. 1999. Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1. Virology 255:347-353), and humans (Kapusta, J., M. Modelska, M. Figlerowicz, T. Pniewski, M. Letellier, O. Lisowa, V. Yusibov, H. Koprowski, A. Plucienniczak, A. B. Legocki. 1999. A plant-derived edible vaccine against hepatitis B virus. FASEB J. 13:1796-1799) comparable to that of the original pathogen. Characterization studies of these engineered immunogens have proven the ability of plants to express, fold and modify proteins in a manner that is consistent with the native source.
The utilization of transgenic plants for vaccine production has several potential benefits over traditional vaccines. First, transgenic plants are usually constructed to express only a small antigenic portion of the pathogen or toxin, eliminating the possibility of infection or innate toxicity of the whole organism and reducing the potential for adverse reactions. Second, since there are no known human or animal pathogens that are able to infect plants, concerns with viral or prion contamination are eliminated. Third, immunogen production in transgenic crops relies on the same established technologies to sow, harvest, store, transport, and process the plant as those commonly used for food crops, making transgenic plants a very economical means of large-scale vaccine production. Fourth, expression of immunogens in the natural protein-storage compartments of plants maximizes stability, minimizes the need for refrigeration and keeps transportation and storage costs low (Lamphear, B. J., S. J. Streatfield, J. M. Jilka, C. A. Brooks, D. K. Barker, D. D. Turner, D. E. Delaney, M. Garcia, B. Wiggins, S. L. Woodard, E. E. Hood, I. R. Tizard, B. Lawhorn, J. A. Howard. 2002. Delivery of subunit vaccines in maize seed. J. Control. Release 85:169-180). Fifth, formulation of multicomponent vaccines is possible by blending the seed of multiple transgenic corn lines into a single vaccine. Sixth, direct oral administration is possible when immunogens are expressed in commonly consumed food plants, such as grain, leading to the production of edible vaccines.
Some of the first edible vaccine technologies developed include transgenic potatoes expressing the E. coli heat-labile enterotoxin (LT-B) or a Hepatitis B surface antigen (HbsAg); (Thanavala, supra; Arntzen, C. J., D. M.-K. Lam. 2000. Vaccines expressed in plants. U.S. Pat. No. 6,136,320; Lam, D. M.-K., C. J. Arntzen, H. S. Mason. 2000. Vaccines expressed in plants. U.S. Pat. No. 6,034,298; Arntzen, C. J., D. M.-K. Lam. 1999. Vaccines expressed in plants. U.S. Pat. No. 5,914,123; Lam, D. M.-K., C. J. Arntzen. 1997. Anti-viral vaccines expressed in plants. U.S. Pat. No. 5,612,487; Lam, D. M., C. J. Arntzen. 1996. Vaccines produced and administered through edible plants. U.S. Pat. No. 5,484,719), and a Norwalk virus surface protein (Mason, 1996, supra). In addition to human viral targets, two proteins specific for livestock viruses have also been expressed in plants and fed to animals to test for immune responses, VP1 protein for foot-and-mouth disease (Wigdorovitz, supra; Carillo, C., A. Wigdorovitz, J. C. Oliveros, P. I. Zamorano, A. M. Sadir, N. Gomez, J. Salinas, J. M. Escribano, M. V. Borca. 1998. Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants. J. Virology 72:1688-1690) and Transmissable Gasteroenteritis Virus (Jilka, J. Immunogenicity of TGEV spike protein expressed in transgenic maize seed: preliminary swine trials. PCT/US01/01148)
One of the most promising aspects of edible vaccines is the ability of orally administered immunogens to stimulate a mucosal immune response (Ruedl, C. and H. Wolf. 1995. Features of oral immunization. Int. Arch. Allergy Immunol. 108:334-339). Mucosal surfaces, the linings of the respiratory, gastrointestinal, and urogenital tracts, play an important physical and chemical role in protecting the body from invading pathogens and harmful molecules. The mucosal immune system is distinct and independent of the systemic, or humoral, immune system, and is not effectively stimulated by parenteral administration of immunogens (Czerkinsky, C., A. M. Svennerholm, and J. Holmgren. 1993. Induction and assessment of immunity at enteromucosal surfaces in humans: implications for vaccine development. Clin. Infect. Dis. 16 Suppl 2:S106-S116). Rather, the mucosal immune system requires antigen presentation directly upon the mucosal surfaces (Jilka, J. Immunogenicity of TGEV spike protein expressed in transgenic maize seed: preliminary swine trials. WO 01/51080; Bailey, M. R. 2000. A model system for edible vaccination using recombinant avidin produced in corn seed. M. S. degree thesis, Texas A&M University). Since most invading pathogens first encounter one or more of the mucosal surfaces, stimulation of the mucosal immune system is often the best first defense against many transmissible diseases entering the body through oral, respiratory and urogenital routes (Holmgren, J., C. Czerkinsky, N. Lycke, and A. M. Svennerholm. 1994. Strategies for the induction of immune responses at mucosal surfaces making use of cholera toxin B subunit as immunogen, carrier, and adjuvant. Am. J. Trop. Med. Hyg. 50:42-54).
Diabetes mellitus is a serious and chronic disorder that affects 6% of the world's population and all ethnic groups. In the United States, approximately 5% of the population has diabetes. Symptoms of diabetes include hyperglycemia and reduced production or release of insulin or response to insulin. Diabetes mellitus is classified into two types, type I diabetes or insulin-dependent diabetes mellitus (IDDM) and type II diabetes or non-insulin-dependent diabetes mellitus (NIDDM). Type I diabetes, in which the pancreas has stopped producing insulin, affects 10% of all diabetics, often begins in childhood and is known as juvenile onset diabetes. Significant recent research has focused on the development of molecules for treatment of insulin-dependant diabetes mellitus. This autoimmune disease results in the destruction of insulin-producing pancreatic cells. Eisenbarth, G. (1985) N. Eng. J. Med 314:1360-1388. In the more prevalent type II diabetes, affecting 90% of all diabetics, the pancreas can produce insulin, but insulin secretion in response to meals is diminished, and the diabetic's tissues, are not as responsive to insulin as tissues from a non-diabetic. Type II diabetes is also known as adult onset diabetes.
Diminished response to or low levels of insulin result in chronic high levels of blood glucose, which gradually alters normal body chemistry and leads to failure of the microvascular system in many organs. This leads to dire consequences. For example, in the United States, diabetes is the largest cause of blindness, is involved in about 70% of amputations, and is the cause of kidney failure in 33% of patients requiring dialysis. Medical treatment of side effects of diabetes and lost productivity due to inadequate treatment of diabetes are estimated to have an annual cost of about $40 billion in the United States alone.
It has long been a goal of insulin therapy to mimic the pattern of endogenous insulin secretion in normal individuals. The daily physiological demand for insulin fluctuates and can be separated into two phases: (a) the absorptive phase requiring a pulse of insulin to dispose of the meal-related blood glucose surge, and (b) the post-absorptive phase requiring a sustained delivery of insulin to regulate hepatic glucose output for maintaining optimal fasting blood glucose. Accordingly, effective therapy for people with diabetes generally involves the combined use of two types of exogenous insulin formulations: a fast-acting meal time insulin provided by bolus injections and a long-acting, so-called, basal insulin, administered by injection once or twice daily to control blood glucose levels between meals.
The nine-year Diabetes Control and Complications Trial (DCCT), which involved 1441 type I diabetic patients, demonstrated that maintaining blood glucose levels within close tolerances reduces the frequency and severity of diabetes complications. Conventional insulin therapy involves only two injections per day. The intensive insulin therapy in the DCCT study involved three or more injections of insulin each day. In this study the incidence of diabetes-side effects was dramatically reduced. For example, retinopathy was reduced by 50-76%, nephropathy by 35-56%, and neuropathy by 60% in patients employing intensive therapy.
Unfortunately, many diabetics are unwilling to undertake intensive therapy due to the discomfort associated with the many injections required to maintain close control of glucose levels. A non-injectable form of insulin is desirable for increasing patient compliance with intensive insulin therapy and lowering their risk of complications. Many investigators have studied alternate routes for administering insulin, such as oral, rectal, transdermal, and nasal routes. So far, these types of administration have not been effective due to poor insulin absorption, low serum insulin concentration, irritation at the site of delivery, or lack of significant decrease in serum glucose levels. Therefore, there remains a need for an effective system for administration of a long-acting insulin.
The source of most insulin today is isolation from human, bovine or pork tissue or serum, or from recombinant microorganisms; yeast or the bacteria E. coli. Demand for the compound is huge. When attempts have been made to provide non-injection routes of insulin administration, the amount required increases further. Currently, insulin is a three billion to five billion dollar market. Several metric tons are provided yearly. With non-injection methods, volume is predicted to increase ten times. Clearly, any reduction in cost would be advantageous, along with the ability to produce large quantities in an easily transported and stored manner. Still further, an oral delivery method would provide a highly desirable alternative to current choices of administering insulin.
With adequate expression levels, large quantities of proinsulin or insulin could be produced, very economically, for pharmaceutical and other uses. Insulin is also useful, in addition to its role in treatment of diabetics, for research and related purposes. For example, in addition to its role in vivo, insulin is also useful in recombinant cell culture. Insulin is an example of a polypeptide factor important for mammalian cell culture proliferation and anabolism. Some cell cultures produce endogenous insulin and some do not; cell cultures which rely on added insulin are problematic because insulin is unstable in some cultures. European publication number, 0307247A2, published Mar. 3, 1989, describes the introduction of nucleic acid encoding insulin into a mammalian host cell to eliminate the need for adding exogenous insulin.
Production of insulin in plants would overcome problems associated with current production methods, such as the limited amount of insulin and proinsulin which is available from animal and bacteria and yeast sources, and purity issues. Attempts have been made to express insulin and another protein associated with diabetes, glutamate decarboxylase, in plants. Indeed, initial attempts did not use the insulin or proinsulin gene, but rather the diabetes-associated autoantigen, glutamic acid decarboxylase was expressed in potato and tobacco. Ma et al., Nature Medicine (1997) 3(7):793-796. Also, potato plants were transformed with the proinsulin gene fused to cholera toxin B subunit as discussed in Arakawa et al. Nature Biotechnology (1998) 16:934-938. This resulted in the fusion protein being produced at up to 0.05% of total soluble protein. However, fusion of insulin with the cholera toxin B subunit may not always be desirable. These experiments were conducted with dicotyledonous plants. The inventors have discovered it is not necessary to fuse insulin with cholera toxin B subunit in order to achieve expression.